CN105567685B - AaPDR3 gene promoters and its application - Google Patents
AaPDR3 gene promoters and its application Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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Abstract
The invention discloses a kind of promoter, which is energy controlling gene specifically expressing in nonsecreting type glandular hairs, nucleotide sequence such as SEQ ID No：It is the promoter of AaPDR3 genes shown in 1.The promoter be can be applied to using plant nonsecreting type glandular hairs tissue expression and be produced in the genetic engineering breeding of metabolite.In addition, the invention also discloses carriers and expression cassette comprising the promoter.The present invention is also generation and the metabolic regulation of development and secondary metabolite therein of further investigation nonsecreting type glandular hairs, and carrying out research especially by the promoter proAaPDR3 of AaPDR3 genes lays a good foundation.
Description
Technical field
The invention belongs to bioengineering field, it is related to a kind of promoter and its application, more particularly to a kind of overstepping one's bounds in plant
Secrete specifically expressed transporter gene promoter (proAaPDR3) in type glandular hairs and its application in transgenic plants.
Background technology
Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia, and plant has strong volatility
Fragrance contains many secondary metabolites in plant：Qinghaosu, volatile oil, australene, camphor, artemisia ketone etc., additionally contain
There is flavone compound.Currently, qinghaosu conjoint therapy (Artemisinin-based combination therapies,
ACTs) be world health organisation recommendations most effective treatment malaria method.But content of its qinghaosu in plant ginghao
It is low, it there is no method to fully meet the market demand in the whole world.
Sweet wormwood has secreting type glandular hairs (glandular trichomes) and nonsecreting type glandular hairs (nonglandular
trichomes).It in the front and back of sweet wormwood blade, stalk, takes and all largely there are glandular hairs, be a large amount of secondary metabolisms here
The accumulation place of object, qinghaosu are considered being stored in secreting type glandular hairs.Two kinds of glandular hairs are for the growth of plant, development, defence, flower
Powder propagation etc. is all played the role of vital.
Promoter is the specific nucleic acid sequence for being located at structural gene 5 ' and holding upstream, can regulate and control the expression of downstream gene, open
Mover plays its specific work(just as one " switch " by the interaction of cis-acting elements and trans-acting factor
Energy.Promoter one is divided into three kinds：Constitutive promoter, specific promoter, inducible promoter.In sweet wormwood genetic engineering research
In, most of currently used promoter is the promoter of composition.This kind of promoter can drive gene all in plant
Tissue and organ in express, the waste of plant intracellular metabolite can be led to, it is also possible to be caused to the normal growth of plant certain
Burden and harm.And the specifically expressed promoter of nonsecreting type glandular hairs only carries out hereditary behaviour to the nonsecreting type glandular hairs system of plant
Make, the waste of metabolism and growth and development will not be caused to cause damages to plant.
ABC (ATP binding cassette) transport protein is a major class greatly very special super family
Race.Most of abc transport albumen participates directly in the transdermal delivery of various molecules.AaPDR3 is one cloned from sweet wormwood
The transport protein of PDR (Pleiotropic drug resistance) subfamily in a abc transport albumen.
Therefore, those skilled in the art are dedicated to developing a kind of promoter of the specifically expressed AaPDR3 genes of glandular hairs.
Invention content
Gene can be driven to be expressed in plant all tissue and organ in view of promoter in the prior art, can caused
The waste of plant intracellular metabolite, it is also possible to which certain burden and these defects of harm, this hair are caused to the normal growth of plant
It is bright that the technical problem to be solved is that provide one kind specifically expressed promoter in plant nonsecreting type glandular hairs, energy guiding gene
The specifically expressing in genetically modified plants nonsecreting type glandular hairs.
To achieve the above object, an aspect of of the present present invention provides a kind of promoter, which is that energy controlling gene exists
Specifically expressing in nonsecreting type glandular hairs, nucleotide sequence such as SEQ ID No：Shown in 1.The promoter opens for transporter gene
Mover is inducible promoter and specific promoter.
Further, which is the promoter of sweet wormwood AaPDR3 upstream region of gene.
Further, the cis-acting elements in the promoter includes：TATA box、CAAT box、G-box、AE-box、
ARE, GA-motif, G-box, MBS and W box.
Another aspect of the present invention provides the application of above-mentioned promoter, is to utilize plant nonsecreting type glandular hairs tissue expression
With the application in the genetic engineering breeding of production metabolite.
Further, wherein metabolite is qinghaosu.
Another aspect of the present invention provides a kind of carrier, which is connected with promoter as described above.The carrier can be with
It is pCAMBIA1391z carriers.
Another aspect of the invention provides a kind of method for the transgene abrotanum preparing high yield qinghaosu, including following step
Suddenly：
Step 1: according to AaPDR3 gene promoter sequences in sweet wormwood genome, nest-type PRC clone such as claim are utilized
The sequence of promoter described in 1；
It is carried Step 2: the sequence of the promoter of the AaPDR3 genes obtained in step 1 is connected into pCAMBIA1391z
Body obtains pCAMBIA1391Z-proAaPDR3 carriers；
Step 3: the pCAMBIA1391Z-proAaPDR3 carriers obtained in step 2 are converted Agrobacterium tumefaciems；
Step 4: the Agrobacterium tumefaciens transformation with pCAMBIA1391Z-proAaPDR3 carriers that will be obtained in step 3
Sweet wormwood, and be detected；
Step 5: determining the expressive site of gus reporter gene that the promoter is guided in plant.
Preferably, wherein promoter is merged with the key gene in artemisinin synthesis approach, to reinforce sweet wormwood synthesis
Approach, the final synthesis for improving qinghaosu.Key gene in the artemisinin synthesis approach can be PDR3 genes etc..
Preferably, the primer sequence of wherein nest-type PRC is：
First round PCR：PF1：5’-CTTTTCCAGCCAAATAAGTATGCCG-3’；
PR1：5’-TTCTTCCACTATTGCTCCCTAACCT-3’；
Second wheel PCR：PF2：5’-TTTCTTTGGTGTTGTATCAATCTCTG-3’；
PR2：5’-TTCTTCCACTATTGCTCCCTAACCT-3’；
Preferably, to build pCAMBIA1391z-proAaPDR3 carriers, the both ends proAaPDR3 introduce EcoRI respectively
Restriction enzyme site and NcoI restriction enzyme site, the primer sequence used is：
EcoRI-Pro-PDR3-FP：5’-CGGAATTCGAACTGTTGAATTAGTATTTAC-3’；
Pro-PDR3-NcoI-RP：5’-CATGCCATGGTTTTAATGCTCAAAAAGCCC-3’.
The present invention also provides a kind of expression cassette, which includes foregoing promoter.Preferably, the expression cassette
For proAaPDR3-GUS expression cassettes.Further, in the expression cassette, the key enzyme in artemisinin synthesis approach can be inserted
Gene.
Compared with prior art, the beneficial effects of the invention are as follows：The present invention is special using effective sweet wormwood epidermis glandular hairs tissue
Specific Promoters replace constitutive promoter, can be built by the method for molecular biology special in sweet wormwood nonsecreting type glandular hairs
The fusion for expressing target gene, is transferred in the genome of other plant using genetic transfoumation, to realize mesh
The directional operation of gene obtained genetically modified plants, and will not cause damages, can be widely used in the growth and development of plant
Using in the genetic engineering breeding of plant glandular hairs tissue expression and production metabolite.In addition, the present invention is also that further investigation is non-
The generation and development of secreting type glandular hairs and the metabolic regulation of secondary metabolite therein, especially by AaPDR3 genes
Promoter proAaPDR3 carries out research and lays a good foundation.
The technique effect of the design of the present invention, specific steps and generation is described further below with reference to attached drawing, with
It is fully understood from the purpose of the present invention, feature and effect.
Description of the drawings
Fig. 1 is the PCR testing result figures of the transgene abrotanum plant of the preferred embodiment of the present invention.Wherein, M：Point
Son amount label；Swimming lane 1：Negative control；Swimming lane 2：Positive control；Swimming lane 3-12：It is 10 plants of transgene abrotanum plant genes respectively
Group is masterplate, carries out the product that PCR is obtained.
Fig. 2 is that the preferred embodiment of the present invention is merged using AaPDR3 gene promoters proAaPDR3 with gus gene
Carrier pCAMBIA1391z-proAaPDR3 conversion sweet wormwood after, the GUS tissue staining figures in the transgene abrotanum obtained.
Specific implementation mode
Test method without specific conditions in following embodiments, usually according to normal condition, such as Sambrook etc.
People's《Molecular cloning：Laboratory manual》(New York：Cold Spring Harbor Laboratory Press, 1989
Version) described in condition, or according to the normal condition proposed by manufacturer.
The Agrobacterium tumefaciems EHA105 that the present embodiment is related to exists《Huang Yali, Jiang Xiliang, field Yunlong, Guo Ping, Zhu Changxiong；
The research of Agrobacterium tumefaciens mediated trichoderma harzianum genetic transformation, Chinese biological engineering magazine, 2008,28 (3)：38-43》Text
Offer middle disclosure.Agrobacterium tumefaciems EHA105 and plasmid pCAMBIA1391z can be obtained by disclosing commercially available commercial channel, such as may be used
To be bought from Australian CAMBIA companies, strain number Gambar1.
The acquisition of 1 AaPDR3 gene promoters proAaPDR3 of embodiment
1, sweet wormwood aseptic seedling is cultivated
The ethyl alcohol that seeds of southernwood volume fraction is 75% impregnates 1min, then impregnates 20min with the NaClO of 20% (w/v)
Afterwards, aseptic water washing 3 times -4 times are blotted surface moisture with sterile blotting paper, are inoculated on the MS solid mediums of no hormone, 25
DEG C, 16h/8h (daylight/night) illumination cultivation, sweet wormwood aseptic seedling is can be obtained after 14 days.
2, the extraction of genomic DNA
A piece of sweet wormwood blade (1cm is put in 1.5mL centrifuge tubes2Left and right size is taken with ice chest Sheng), 2 steel balls are added.Add
Enter 300 μ L TPS buffer (being operated in draught cupboard, contain mercaptoethanol in TPS), 55-60Hz shakes 90 seconds.Add 300
μ L TPS buffer (draught cupboard).65 DEG C of water-bath 1h (being shaken every 20min), can be appropriately extended time, longest 1.5h.It is cold
But to room temperature, 4 DEG C of 10000rpm centrifuge 15min.Take 300 μ L-400 μ L supernatants.300 μ L-400 μ L isopropanol (isopropyls are added
Alcohol is pre-chilled at -20 DEG C).10-15min (1h can be extended to) is placed after mixing in -20 DEG C of refrigerators.Take out 4 DEG C of centrifugations
12000rpm sucks supernatant, and 10-15min is inverted in draught cupboard.75% ethyl alcohol 500-600 μ L are added, will precipitation blown and beaten or
It is bounced with finger, is placed on shaking table and shakes 15-20min, be repeated once.Liquid is blotted, is placed in 37 DEG C and dries, until precipitation becomes
It is transparent.50 μ L ddH are added2O back dissolvings obtain genomic DNA, 4 DEG C of preservations.
3, PCR amplification
Using the genomic DNA of above-mentioned acquisition as template, nonsecreting type glandular hairs specific promoter is expanded using PCR method
ProAaPDR3 sequences.In order to improve the specificity of product, using two-wheeled nested PCR amplification, obtained according to gene order-checking
The promoter sequence design nest-type PRC primer of AaPDR3 genes is as shown in table 1, and first round PCR is shown in reaction system such as table 2.PCR
Condition is：94 DEG C of pre-degeneration 10min；94 DEG C of 40s, 50 DEG C of 40s, 68 DEG C of 3min, 34 cycles；68 DEG C of extension 10min.1%
Ago-Gel in electrophoresis detection PCR product.
1 nest-type PRC primer of table
2 first round of table PCR reaction systems
The product dilution 50 of first round PCR is used as to the masterplate of the second wheel PCR again, the primer of the second wheel PCR be FP2 and
RP2, reaction system is as shown in table 3, and PCR reaction conditions are：94 DEG C of pre-degeneration 10min；94 DEG C of 40s, 55 DEG C of 40s, 68 DEG C of 2min,
34 cycles；68 DEG C of extension 10min.PCR product is detected with 1% agarose gel electrophoresis, is recycled specific band, is connected to
PJET1.2 carriers are for being sequenced.The sequence of the segment and the sequence of AaPDR3 genes are spliced, AaPDR3 is as a result obtained
The sequence of upstream region of gene about 2kb.The sequence such as SEQ ID No of the AaPDR3 gene promoters proAaPDR3 of acquisition：Shown in 1.
Table 3 second takes turns PCR reaction systems
Embodiment 2 analyzes the cis-acting elements of promoter proAaPDR3, determines the type of proAaPDR3
The promoter proAaPDR3 sequence lengths obtained in embodiment 1 are 2059bp.It is suitable above promoter in order to find
Formula functional element, with Plantcare (http://bioinformatics.psb.ugent.be/webtools/plantcare/
Html/) promoter proAaPDR3 is analyzed.Analysis finds have in addition to TATA box above the polyclonal promoter arrived
With CAAT box, also there are many cis-acting elements, such as：G-box, AE-box, ARE, GA-motif, G-box, MBS and W
Box etc., it is specific as shown in table 4.Wherein, G-box is found in many plant promoters, it is that many responses start
Son functions necessary element, is played very in plant promoter is to the response process of light, oxygen-free environment and plant hormone
Important role；In addition, GA-motif light regulates and controls, ABRE can respond abscisic acid in plant；MBS is myb transcription factor
Binding site.Result above analysis shows, the promoter proAaPDR3 of AaPDR3 genes is the promoter of an induction type, energy
It is induced by many factors.
Controlling element is analyzed in 4 promoter proAaPDR3 sequences of table
AaPDR3 gene promoters proAaPDR3 is connected into pCAMBIA-1391z carriers, fusion GUS reports by embodiment 3
Gene
In order to study expression of the gene promoter in plant different tissues position, by the promoter of AaPDR3 genes
ProAaPDR3 connection pCAMBIA-1391z carriers, to merge gus reporter gene.In order to realize the structure to expression vector,
When being expanded to promoter proAaPDR3, EcoRI restriction enzyme sites are introduced by forward primer, by drawing in reverse primer
Enter NcoI restriction enzyme sites, primer sequence is as shown in table 5：
The PCR primer that 5 pCAMBIA1391z-proAaPDR3 vector constructions of table use
By double digestion and the connection of proAaPDR3 segments and pCAMBIA-1391z carriers to amplification, obtain
PCAMBIA1391z-proAaPDR3 carriers.
The pCAMBIA1391z-proAaPDR3 carriers built are converted Agrobacterium tumefaciems and detected by embodiment 4
The plant expression vector pCAMBIA1391z-proAaPDR3 completed containing structure is transferred to Agrobacterium tumefaciems
(EHA105), and PCR verifications are carried out.The result shows that：Plant expression vector pCAMBIA1391z- containing promoter gene fragment
ProAaPDR3 is successfully building up in Agrobacterium tumefaciens strain, to obtain the plant merged containing gene promoter and gus gene
The Agrobacterium tumefaciens strain of expression vector pCAMBIA1391z-proAaPDR3.
Embodiment 5 is by the Agrobacterium tumefaciens transformation sweet wormwood with pCAMBIA1391z-proAaPDR3 carriers and examines
It surveys
1) preculture of explant
The ethyl alcohol that seeds of southernwood volume fraction is 75% impregnates 1min；Again 20min is impregnated with the NaClO of 20% (w/v)；
Aseptic water washing 3-4 times；Surface moisture is blotted with sterile blotting paper；It is inoculated in the MS culture mediums of no hormone, 25 DEG C, 16 hours
Daylight and 8 hours night culture, you can obtain sweet wormwood aseptic seedling.After seedling is grown to 5cm or so, outside clip tests for sterility
Implant is for converting.
Wherein, MS culture mediums are the solid medium that Murashige and Skoog was invented in 1962, the solid medium
It can be obtained by commercial channel.
2) co-cultivation of Agrobacterium and explant
By the blade explant, the co-cultivation training of 1/2MS and 100 μm of ol/L AS (acetosyringone) composition is gone to
It supports in base, the activated Agrobacterium tumefaciems engineering bacteria containing pCAMBIA1391z-proAaPDR3 plant expression vectors is added dropwise
1/2MS suspensions, make explant be come into full contact with bacterium solution, 28 DEG C of light cultures 3 days.The crown gall agriculture bar without target gene is added dropwise
The blade explant of the 1/2MS fluid nutrient medium suspensions of bacterium is control.
3) screening and detection of resistance regeneration plant
By the sweet wormwood explant of the co-cultivation 3 days be transferred to MS, 0.5mg/L 6-BA (6- benzyl purines),
The germination sieve of 0.05mg/L NAA (methyl α-naphthyl acetate), 50mg/L Kan (kanamycins) and 500mg/L Cb (carbenicillin) compositions
It selects on culture medium, is cultivated in 25 DEG C, 16 hours daylight and 8 hours dark, squamous subculture is primary every two weeks, by 2-3
It can be obtained Kan resistance Multiple Buds after secondary subculture.Well-grown resistance Multiple Buds are cut and are transferred to 1/2MS0(the MS of no hormone
Culture medium) and the root media of 125mg/L Cb composition on cultivate to taking root, regenerate sweet wormwood plant to obtain Kan resistances.
Forward primer is separately designed according to proAaPDR3 the and GUS sequences in expression cassette proAaPDR3-GUS sequences
(proPDR3-F:5 '-TGAAATTACTATATAATTAGCATAG-3 ') and reverse primer (GUS-R:5’-
GACATCGGCTTCAAATGGCGTA-3 ') gus gene is detected.Using designed PCR special primers, to picking
10 plants of transfer-gen plants carry out PCR detections respectively.The result shows that wherein 8 plants can amplify specific DNA fragment (i.e. gus gene portion
Fragment section), as shown in Figure 1.
The determination of expressive site of the gus reporter gene that 6 promoter proAaPDR3 of embodiment is guided in plant
To the sweet wormwood plant of PCR test positive, its blade is taken to carry out GUS tissue stainings, the results are shown in Figure 2, wherein
Dyeing part is specific expressed in the nonsecreting type glandular hairs of sweet wormwood, illustrates that AaPDR3 gene promoters proAaPDR3 is turning base
Because foreign gene specifically expressing in glandular hairs can be guided in sweet wormwood.It can be seen that the proAaPDR3 genes that the present invention is cloned
Promoter can be used for using plant glandular hairs tissue expression and produce in the genetic engineering breeding and industrialization of metabolite.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (8)
1. a kind of promoter, which is characterized in that the promoter is energy controlling gene specifically expressing in nonsecreting type glandular hairs,
Nucleotide sequence such as SEQ ID No：Shown in 1.
2. the application of promoter as described in claim 1, which is characterized in that utilizing plant nonsecreting type glandular hairs tissue expression
With the application in the genetic engineering breeding of production metabolite；The plant is sweet wormwood.
3. application as claimed in claim 2, which is characterized in that the metabolite is qinghaosu.
4. a kind of carrier, which is characterized in that the carrier is connected with promoter as described in claim 1.
5. a kind of method for the transgene abrotanum preparing high yield qinghaosu, which is characterized in that include the following steps：
Step 1: according to AaPDR3 gene promoter sequences in sweet wormwood genome, using nest-type PRC clone as in claim 1
The sequence of the promoter；
Step 2: the sequence of the promoter of the AaPDR3 genes obtained in step 1 is connected into pCAMBIA1391z carriers,
Obtain pCAMBIA1391Z-proAaPDR3 carriers；
Step 3: the pCAMBIA1391Z-proAaPDR3 carriers obtained in step 2 are converted Agrobacterium tumefaciems；
Step 4: the Agrobacterium tumefaciens transformation with pCAMBIA1391Z-proAaPDR3 carriers obtained in step 3 is green
Wormwood artemisia, and be detected；
Step 5: determining the expressive site of gus reporter gene that the promoter is guided in plant.
6. the method for the transgene abrotanum according to claim 5 for preparing high yield qinghaosu, which is characterized in that the startup
Son is merged with the key gene in artemisinin synthesis approach.
7. the method for the transgene abrotanum according to claim 5 for preparing high yield qinghaosu, which is characterized in that the nido
The primer sequence of PCR is：
First round PCR：PF1：5’-CTTTTCCAGCCAAATAAGTATGCCG-3’；
PR1：5’-TTCTTCCACTATTGCTCCCTAACCT-3’；
Second wheel PCR：PF2：5’-TTTCTTTGGTGTTGTATCAATCTCTG-3’；
PR2：5’-TTCTTCCACTATTGCTCCCTAACCT-3’；
For build pCAMBIA1391z-proAaPDR3 carriers, the both ends proAaPDR3 introduce respectively EcoRI restriction enzyme site and
The restriction enzyme site of NcoI, the primer sequence used are：
EcoRI-Pro-PDR3-FP：5’-CGGAATTCGAACTGTTGAATTAGTATTTAC-3’；
Pro-PDR3-NcoI-RP：5’-CATGCCATGGTTTTAATGCTCAAAAAGCCC-3’.
8. a kind of expression cassette, which is characterized in that the expression cassette includes promoter as described in claim 1.
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| WO2007005604A2 (en) * | 2005-07-05 | 2007-01-11 | The Regents Of The University Of California | Polynucleotides encoding isoprenoid modifying enzymes and methods of use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007005604A2 (en) * | 2005-07-05 | 2007-01-11 | The Regents Of The University Of California | Polynucleotides encoding isoprenoid modifying enzymes and methods of use thereof |
| CN105087579A (en) * | 2015-07-13 | 2015-11-25 | 上海交通大学 | Promoter for regulating expression of genes in T shape trichome and application of promoter |
Non-Patent Citations (1)
| Title |
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| 青蒿素的合成生物学研究进展;孔建强 等;《药学学报》;20131231;第48卷(第2期);第193-205页 * |
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